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J. Biol. Chem., Vol. 277, Issue 4, 2750-2755, January 25, 2002

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Functional Analysis of Tryptophans 62 and 120 on HLA-DM^*

Amélie Faubert^§, Angela Samaan^¶, and Jacques Thibodeau

From the  Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada and ^¶ INSERM U462, Hopital Saint-Louis, 75475, Paris, France

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM W62A,W120A (DM_W62A/W120A) double mutant was expressed in HLA-DR⁺ HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DM_W62A/W120A removes CLIP as efficiently as its wild-type counterpart. DM_W62A/W120A was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations W62A and W120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DM_W62A/W120A were co-immunoprecipitated at pH 7. We conclude that the 62 and 120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.

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